Transcriptome analysis of Atlantic Herring (Clupea harengus) using Next Generation Sequencing (NGS)

The Atlantic herring is one of the most abundant fish species in the Northern hemisphere especially in the Northeast Atlantic. There are various stocks of this fish due to their spawning time and their vast distribution. Many studies have tried to characterize herring populations and these efforts can be combined with massively parallel sequencing technologies to develop genetic resources. The transcriptome is a repertoire of RNAs in cells produced by transcription and messenger RNAs (mRNAs) are responsible for gene expression. This repertoire may change during different life stages and environmental conditions. Transcriptome studies have been mainly dependent on a reference genome and developed tools are not applicable for non-model organisms for which the reference genome is missing or only partially available. In this study we present a de novo transcriptome assembly by using different strategies and one specific transcriptome assembler, Trinity. The RNA was collected from muscle of a male spring spawning herring and then sequenced with an Illumina Hiseq 2000 machine. After trimming for low quality nucleotides and possible contaminations, the assembly resulted in 131,788 contigs with a total size of 40 million nucleotides (Mnts). The transcriptome generated by Trinity was compared with other assemblies, assembled by a genome assembler (SOAPdenovo), Inchworm, SSPACE, and CAP3. The results showed that Trinity developed a more reliable assembly. We validated almost 47% of all contigs by comparison to available databases such as nr and other vertebrate transcripts. Also, we quantified the relative expression of transcripts by counting the number of aligned reads per kilobase per million mapped reads (RPKM). Furthermore, we preformed indepth studies of two genes. We identified two copies of Glucose 6-phosphate isomerase (GPI) on draft genome assembly. Our results showed that the sampled fish was heterozygous at the GPIb locus. Among the alpha actin isoforms, we identified the alpha actin b transcript (ACTA1b) in fast muscle corresponding to spring spawning herring fish characteristics

Dangerous women of Hong Kong? Media construction of stigma in female sex workers

This study used a cultural model analysis to examine the Hong Kong print media’s social construction of stigma in respect to female sex workers. An analysis was conducted on captions and main headlines of two newspaper (Chinese and English) median in Hong Kong, 2003-2006. A total of 591 articles on sex workers were recruited in the analysis with 422 located from the Ming Pao and 169 articles the SCMP. A total of Sixty seven articles on health issues were identified. In Hong Kong, as in elsewhere, sex workers were commonly labeled as the sources of sexually transmitted diseases and as women who endangered the public safety through socially unacceptable occupations. They were also portrayed as “ugly”, “weak” and “powerless” in the articles identified. We conclude the Hong Kong print media plays a significant role in contributing to the stigmatization of sex workers, heightening health risk and vulnerability. Such social construction of public stigma then in turn, can be argued to contribute to a lessened propensity for female sex workers both seek and engage with formal health services.published_or_final_versio

Whole-genome genotyping and resequencing reveal the association of a deletion in the complex interferon alpha gene cluster with hypothyroidism in dogs

Background: Hypothyroidism is a common complex endocrinopathy that typically has an autoimmune etiology, and it affects both humans and dogs. Genetic and environmental factors are both known to play important roles in the disease development. In this study, we sought to identify the genetic risk factors potentially involved in the susceptibility to the disease in the high-risk Giant Schnauzer dog breed. Results: By employing genome-wide association followed by fine-mapping (top variant p-value=5.7x10(-6)), integrated with whole-genome resequencing and copy number variation analysis, we detected a similar to 8.9 kbp deletion strongly associated (p-value=0.0001) with protection against development of hypothyroidism. The deletion is located between two predicted Interferon alpha (IFNA) genes and it may eliminate functional elements potentially involved in the transcriptional regulation of these genes. Remarkably, type I IFNs have been extensively associated to human autoimmune hypothyroidism and general autoimmunity. Nonetheless, the extreme genomic complexity of the associated region on CFA11 warrants further long-read sequencing and annotation efforts in order to ascribe functions to the identified deletion and to characterize the canine IFNA gene cluster in more detail. Conclusions: Our results expand the current knowledge on genetic determinants of canine hypothyroidism by revealing a significant link with the human counterpart disease, potentially translating into better diagnostic tools across species, and may contribute to improved canine breeding strategies

Comparative omics and feeding manipulations in chicken indicate a shift of the endocrine role of visceral fat towards reproduction.

BACKGROUND: The mammalian adipose tissue plays a central role in energy-balance control, whereas the avian visceral fat hardly expresses leptin, the key adipokine in mammals. Therefore, to assess the endocrine role of adipose tissue in birds, we compared the transcriptome and proteome between two metabolically different types of chickens, broilers and layers, bred towards efficient meat and egg production, respectively. RESULTS: Broilers and layer hens, grown up to sexual maturation under free-feeding conditions, differed 4.0-fold in weight and 1.6-fold in ovarian-follicle counts, yet the relative accumulation of visceral fat was comparable. RNA-seq and mass-spectrometry (MS) analyses of visceral fat revealed differentially expressed genes between broilers and layers, 1106 at the mRNA level (FDR ≤ 0.05), and 203 at the protein level (P ≤ 0.05). In broilers, Ingenuity Pathway Analysis revealed activation of the PTEN-pathway, and in layers increased response to external signals. The expression pattern of genes encoding fat-secreted proteins in broilers and layers was characterized in the RNA-seq and MS data, as well as by qPCR on visceral fat under free feeding and 24 h-feed deprivation. This characterization was expanded using available RNA-seq data of tissues from red junglefowl, and of visceral fat from broilers of different types. These comparisons revealed expression of new adipokines and secreted proteins (LCAT, LECT2, SERPINE2, SFTP1, ZP1, ZP3, APOV1, VTG1 and VTG2) at the mRNA and/or protein levels, with dynamic gene expression patterns in the selected chicken lines (except for ZP1; FDR/P ≤ 0.05) and feed deprivation (NAMPT, SFTPA1 and ZP3) (P ≤ 0.05). In contrast, some of the most prominent adipokines in mammals, leptin, TNF, IFNG, and IL6 were expressed at a low level (FPKM/RPKM< 1) and did not show differential mRNA expression neither between broiler and layer lines nor between fed vs. feed-deprived chickens. CONCLUSIONS: Our study revealed that RNA and protein expression in visceral fat changes with selective breeding, suggesting endocrine roles of visceral fat in the selected phenotypes. In comparison to gene expression in visceral fat of mammals, our findings points to a more direct cross talk of the chicken visceral fat with the reproductive system and lower involvement in the regulation of appetite, inflammation and insulin resistance.The study was supported by the Israel Academy of Sciences grants no. 876/ 14 and 1294/17, and Chief Scientist of the Israeli Ministry of Agriculture 0469/14 (to MFE and ES)

Reconstruction of the birth of a male sex chromosome present in Atlantic herring

The mechanisms underlying sex determination are astonishingly plastic. Particularly the triggers for the molecular machinery, which recalls either the male or female developmental program, are highly variable and have evolved independently and repeatedly. Fish show a huge variety of sex determination systems, including both genetic and environmental triggers. The advent of sex chromosomes is assumed to stabilize genetic sex determination. However, because sex chromosomes are notoriously cluttered with repetitive DNA and pseudogenes, the study of their evolution is hampered. Here we reconstruct the birth of a Y chromosome present in the Atlantic herring. The region is tiny (230 kb) and contains only three intact genes. The candidate male-determining gene BMPR1BBY encodes a truncated form of a BMP1B receptor, which originated by gene duplication and translocation and underwent rapid protein evolution. BMPR1BBY phosphorylates SMADs in the absence of ligand and thus has the potential to induce testis formation. The Y region also contains two genes encoding subunits of the sperm-specific Ca2+ channel CatSper required for male fertility. The herring Y chromosome conforms with a characteristic feature of many sex chromosomes, namely, suppressed recombination between a sex-determining factor and genes that are beneficial for the given sex. However, the herring Y differs from other sex chromosomes in that suppression of recombination is restricted to an similar to 500-kb region harboring the male-specific and sex-associated regions. As a consequence, any degeneration on the herring Y chromosome is restricted to those genes located in the small region affected by suppressed recombination

Exploring genetic diversity in natural and domestic populations through next generation sequencing

Studying genetic diversity in natural and domestic populations is of major importance in evolutionary biology. The recent advent of next generation sequencing (NGS) technologies has dramatically changed the scope of these studies, enabling researchers to study genetic diversity in a whole-genome context. This thesis details examples of studies using NGS data to: (i) characterize evolutionary forces shaping the genome of the Atlantic herring, (ii) detect the genetic basis of speciation and domestication in the rabbit, and, (iii) identify mutations associated with skeletal atavism in Shetland ponies. The Atlantic herring (Clupea harengus) is the most abundant teleost species inhabiting the North Atlantic. Herring has seasonal reproduction and is adapted to a wide range of salinity (3-35‰) throughout the Baltic Sea and Atlantic Ocean. By using NGS data and whole-genome screening of 20 populations, we revealed the underlying genetic architecture for both adaptive features. Our results demonstrated that differentiated genomic regions have evolved by natural selection and genetic drift has played a subordinate role. The European rabbit (Oryctolagus cuniculus) is native to the Iberian Peninsula, where two rabbit subspecies with partial reproductive isolation have evolved. We performed whole genome sequencing to characterize regions of reduced introgression. Our results suggest key role of gene regulation in triggering genetic incompatibilities in the early stages of reproductive isolation. Moreover, we studied gene expression in testis and found misregulation of many genes in backcross progenies that often show impaired male fertility. We also scanned whole genome of wild and domestic populations and identified differentiated regions that were enriched for non-coding conserved elements. Our results indicated that selection has acted on standing genetic variation, particularly targeting genes expressed in the central nervous system. This finding is consistent with the tame behavior present in domestic rabbits, which allows them to survive and reproduce under the stressful non-natural rearing conditions provided by humans. In Shetland ponies, abnormally developed ulnae and fibulae characterize a skeletal deformity known as skeletal atavism. To explore the genetic basis of this disease, we scanned the genome using whole genome resequencing data. We identified two partially overlapping large deletions in the pseudoautosomal region (PAR) of the sex chromosomes that remove the entire coding sequence of the SHOX gene and part of CRLF2 gene. Based on this finding, we developed a diagnostic test that can be used as a tool to eradicate this inherited disease in horses

DNA demethylation regulates gene expression in IgE-activated mouse mast cells

Letter to the Editor

Genomic dynamics of brown trout populations released to a novel environment

Population translocations occur for a variety of reasons, from displacement due to climate change to human-induced transfers. Such actions have adverse effects on genetic variation and understanding their microevolutionary consequences requires monitoring. Here, we return to an experimental release of brown trout (Salmo trutta) in order to monitor the genomic effects of population translocations. In 1979, fish from each of two genetically (F-ST = 0.16) and ecologically separate populations were simultaneously released, at one point in time, to a lake system previously void of brown trout. Here, whole-genome sequencing of pooled DNA (Pool-seq) is used to characterize diversity within and divergence between the introduced populations and fish inhabiting two lakes downstream of the release sites, sampled 30 years later (c. 5 generations). Present results suggest that while extensive hybridization has occurred, the two introduced populations are unequally represented in the lakes downstream of the release sites. One population, which is ecologically resident in its original habitat, mainly contributes to the lake closest to the release site. The other population, migratory in its natal habitat, is genetically more represented in the lake further downstream. Genomic regions putatively under directional selection in the new habitat are identified, where allele frequencies in both established populations are more similar to the introduced population stemming from a resident population than the migratory one. Results suggest that the microevolutionary consequences of population translocations, for example, hybridization and adaptation, can be rapid and that Pool-seq can be used as an initial tool to monitor genome-wide effects

Whole-genome Sequencing of Follicular Thyroid Carcinomas Reveal Recurrent Mutations in MicroRNA Processing Subunit DGCR8

Background: The genomic and transcriptomic landscape of widely invasive follicular thyroid carcinomas (wiFTCs) and Hurthle cell carcinoma (HCC) are poorly characterized, and subsets of these tumors lack information on genetic driver events. Objective: The aim of this study was to bridge this gap. Methods: We performed whole-genome and RNA sequencing and subsequent bioinformatic analyses of 11 wiFTCs and 2 HCCs with a particularly poor prognosis, and matched normal tissue. Results: All wiFTCs exhibited one or several mutations in established thyroid cancer genes, including TERT (n=4), NRAS (n=3), HRAS, KRAS, AKT, PTEN, PIK3CA, MUTYH, TSHR, and MEN1 (n=1 each). MutSig2CV analysis revealed recurrent somatic mutations in FAM72D (n=3, in 2 wiFTCs and in a single HCC), TP53 (n=3, in 2 wiFTCs and a single HCC), and EIF1AX (n=3), with DGCR8 (n=2) as borderline significant. The DGCR8 mutations were recurrent p.E518K missense alterations, known to cause familial multinodular goiter via disruption of microRNA (miRNA) processing. Expression analyses showed reduced DGCR8 messenger RNA expression in FTCs in general, and the 2 DGCR8 mutants displayed a distinct miRNA profile compared to DGCR8 wild-types. Copy number analyses revealed recurrent gains on chromosomes 4, 6, and 10, and fusion gene analyses revealed 27 high-quality events. Both HCCs displayed hyperploidy, which was fairly unusual in the FTC cohort. Based on the transcriptome data, tumors amassed in 2 principal clusters. Conclusion: We describe the genomic and transcriptomic landscape in wiFTCs and HCCs and identify novel recurrent mutations and copy number alterations with possible driver properties and lay the foundation for future studies